Abstract of the Research Plan We will, therefore, rigorously test the hypothesis that vaccine-induced CDS* T cells against a single epitope can control replication of the AIDS virus in the Mamu-B*08 model of HIV Elite Control. We will also extend our previous studies in Mamu-B*OS positive macaques to Mamu-B*17 positive macaques, an animal model of spontaneous control of AIDS virus replication with similarities to HLAB* 57 positive human ECs. We will induce CDS* T cells by vaccination and then determine whether these CDS* T cells can control viral replication after high dose challenge of Indian rhesus macaques with SIVmac239. In the final years of our proposal, we will determine whether effective T-cells express particular TCR and RNA transcripts. Specific Aim I We hypothesize that CD8+ T cells against a single epitope bound by the macaque HLA-B*27 equivalent, Mamu-B*08, can control replication of the AIDS virus. To test this hypothesis we will vaccinate eight Mamu-B*08 positive macaques with a small region of Nef encoding the immunodominant Mamu- B*08-restricted Nef RL10 epitope and then challenge them with a high dose of SIVmac239. We already have preliminary evidence that this vaccine regimen is effective and reduces viral replication after a high dose SIVmac239 challenge. We will follow TCR expression and the ability of vaccine-induced CD8+ T cells to secrete a broad range of cytokines. Specific Aim II We hypothesize that CD8+ T cells against four epitopes bound by the macaque HLA-B*57 equivalent, Mamu-B*17, can control replication of the AIDS virus. We will vaccinate eight Mamu-B*17 positive macaques with small regions of Vif, Nef, and a cryptic open reading frame encoding four CDS* T cell epitopes and challenge them with a high dose of SIVmac239. We will follow TCR expression and the ability of vaccine-induced CD8+ T cells to secrete a broad range of cytokines. Specific Aim III We hypothesize that effective CD8+ T cells express particular RNAs. We will vaccinate eight Mamu-B*08 positive macaques with either Nef RL10 (should a single epitope be sufficient for control-see SAI) or three epitopes (should Nef RL10 not be sufficient) and leave eight Mamu-B*08 positive macaques unvaccinated. These 16 monkeys will be challenged with a single high dose of SIVmac239 and we will follow the outcome. We will analyze RNA expression by illumina sequencing of total RNA and by expression arrays from freshly tetramer-sorted cells, both before challenge (in the vaccinees) and after challenge in all of the animals. We will also follow TCR expression and the ability of Mamu-B*08-restricted CD8+ T cells to secrete a broad range of cytokines. We should then be able to correlate RNA expression, TCR expression or the ability to secrete particular cytokines with outcome on an epitope by epitope basis in both the vaccinees and naive animals. Our group has now conducted many different vaccine and challenge studies involving DNA, SIVmac239ANef, rAd5, rYF and rMVA vectors along with i.r. and i.v. challenges with SIVmac239 and SIVsmE660, and thus should have few problems with the outlined experiments (22-28). We also have extensive experience in both ELISPOT and ICS analysis arid have a comprehensive set of,MHC class I tetramers for Mamu-B*08 and -B*17. Dr. Douek has extensive experience in the analysis of TCR expression. We are therefore confident that we will be able to determine whether vaccine-induced Mamu-B*OS and -B*17- restricted CDS* T cell responses can reduce replication of SIV. We expect to reduce viral replication of SIVmac239 in Mamu-B*08 positive animals given our previous results in Mamu-B*08 positive macaques that were vaccinated with the three immunodominant Mamu-B*08 epitopes. We noted an entirely unexpected change in immunodominance in these last challenge experiments. Here the Nef RL10-specific response became immunodominant and correlated with viral control (Fig. 4). Additionally, Danny Douek's results indicated that public TCR expression by Nef RLIO-specific CDS* T cells correlated with control of viral replication (Fig. 5). We expect, therefore, that animals that have high frequency CDS* T cell responses expressing public TCRs against Nef RL10 after the vaccination phase should be the most efficient at controlling viral replication. We will be able to test this with our correlates analysis. Should our expectations be realized, this would suggest that particular CDS* T ceil populations can control viral replication. Understanding why these CDS* T cells are so efficient would be an important area of investigation in subsequent studies.